LC-MS/MS in Plasma
To analyze TL0901 in complex biological matrices by LC-MS/MS, samples must be precleaned or extracted together with an appropriate internal standard (a 12-mer analog in this case) prior to running. We have found both solid-phase extraction and liquid-liquid extraction (LLE) methods were effective for the extraction of phosphorothiate oligonucleotides. However, LLE produced better and more consistent recovery (>75%).
This method has been fully validated for quantitation of TL0901 in human plasma under good laboratory practice (GLP) conditions, including intra- and inter-day precision and accuracy, selectivity/specificity, metabolite interference, and various stability tests. The validated linear range is from 10.0 to 2,500 ng/mL; both inter-day and intra-day precisions were less than 6%, with a mean accuracy (%bias) of -10% to 1.0%. The assay matrix selectivity was also tested at lower level of quantification in 10 different lots of plasmas, and all stability tests are acceptable per the GLP guideline set forth by FDA.
LC-MS/MS provides an unambiguous, sensitive, and efficient means of detecting and quantifying oligonucleotides in complex biological fluids. We have validated many LC-MS/MS assays for various structured oligonucleotides in various matrices and analyzed thousands of samples in support of both clinical and nonclinical GLP studies around the world. We believe that LC-MS/MS represents the future high-throughput platform for the bioanalysis of oligonucleotide therapeutics.